This proposal focuses on B-cell acute lymphoblastic leukemia (B-ALL). Despite a dramatic increase in cure rates over the past decades, a subset of children with B-ALL continues to have unacceptably high rates of relapse. Leukemias in this high-risk group include Philadelphia chromosome positive (Ph+) cases driven by the BCR-ABL tyrosine kinase, but the majority are Ph- negative. Many high-risk Ph-negative B-ALL cases can now be designated as Ph-like B-ALL due to similar gene expression and activation of tyrosine kinases. Emerging data indicate that Ph-like B-ALL is also prevalent in adolescent and young adult patients. Importantly, Ph-like leukemias show decreased kinase signaling upon treatment with tyrosine kinase inhibitors (TKIs) in vitro and TKI treatment suppresses disease in xenograft models. These findings establish strong rationale for clinical testing of TKIs matched to patient-specific genomic lesions in Ph-like B-ALL. However, TKI resistance develops in many cancers, emphasizing the need to target additional survival mechanisms. Using xenograft models of adult Ph+ B-ALL we have reported that second generation, ATP- competitive inhibitors of mTOR work in synergy with TKIs targeting the BCR-ABL oncogene. Combining mTOR kinase inhibitors (TOR-KIs) with TKIs causes regression of leukemia to a greater extent than TKI alone or TKI plus rapamycin, even in tumors with intrinsic resistance to BCR-ABL kinase inhibitors. mTOR kinase inhibitors are well tolerated at therapeutic doses. Because the TKI/TOR-KI combination works well in Ph+ B-ALL xenografts, we believe similar combinations will be effective in Ph-like B-ALL. This project will test this hypothesis, with the goal of obtaining proof-of- concept data supporting clinical trials of TKI plus TOR-KIs in children, adolescents and young adults with Ph-like B-ALL. We will accomplish this objective using cells from established Ph-like B-ALL xenografts, provided by collaborators. The first specific aim is to evaluate the efficacy of dual pathway inhibition on xenografted Ph-like leukemia samples. Endpoints will be percent leukemia cells in the bone marrow, and fraction of cycling cells in the leukemia versus normal bone marrow cell populations. The second specific aim is to test whether the combination treatments suppress survival signaling in B-ALL cells more profoundly than single treatments. We will address this in part through phosphoflow analysis of cells obtained from bone marrow of xenografted mice. We will also validate an emerging technology, CYTOF, for multiparameter analysis of the phosphoproteome in B-ALL cells from bone marrow of treated mice. These studies may lead to new approaches to prevent relapse in high-risk B-ALL and biomarkers to monitor response to therapy.